Compositions and Methods for Treating Celiac Sprue Disease

ABSTRACT

The invention provides compositions and methods for treating celiac sprue.

CROSS REFERENCE

This application claims priority to U.S. Provisional Application Ser.No. 61/521,899 filed on Aug. 10, 2011, incorporated herein by referencein its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Defense AdvancedResearch Projects Agency (DARPA) grant number HR0011-08-1-0085. Thegovernment has certain rights in the invention.

BACKGROUND

Celiac sprue is a highly prevalent disease in which dietary proteinsfound in wheat, barley and rye products known as ‘glutens’ evoke animmune response in the small intestine of genetically predisposedindividuals. The resulting inflammation can lead to the degradation ofthe villi of the small intestine, impeding the absorption of nutrientsSymptoms can appear in early childhood of later in life, and rangewidely in severity, from diarrhea, fatigue and weight loss to abdominaldistension, anemia, and neurological symptoms. There are currently noeffective therapies for this lifelong disease except the totalelimination of glutens from the diet. Although celiac sprue remainlargely underdiagnosed, its' prevalence in the US and Europe isestimated at 0.5-1.0% of the population. The identification of suitablenaturally-occurring enzymes as oral therapeutics for Celiac disease isdifficult due to the stringent physical and chemical requirements tospecifically and efficiently degrade gluten-derived peptides in theharsh and highly acidic environment of the human digestive tract.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides polypeptides comprisingan amino acid sequence at least 75% identical to an amino acid sequenceaccording to SEQ ID NO:35, wherein

(a) the polypeptide degrades a PQPQLP (SEQ ID NO:34) peptide at pH 4;

(b) residue 278 is Ser, reside 78 is Glu, and residue 82 is Asp; and

(c) the polypeptide comprises an amino acid change from SEQ ID NO:67 atone or more residues selected from the group consisting of 73, 102, 103,104, 130, 165, 168, 169, 172, and 179.

In a second aspect, the present invention provides polypeptidecomprising an amino acid sequence at least 75% identical to an aminoacid sequence according to SEQ ID NO:1, wherein

(a) the polypeptide degrades a PQPQLP (SEQ ID NO:34) peptide at pH 4;

(b) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(c) the polypeptide comprises an amino acid change from SEQ ID NO:33 atone or more resides selected from the group consisting of 119, 262, 291,292, 293, 319, 354, 357, 358, 361, and 368.

In various embodiments of the first and second aspect, the polypeptidecomprises an amino acid sequence at least 85%, 95%, or 100% identical toan amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:35. Inanoter embodiment, the polypeptide, comprises an amino acid sequenceaccording to any one of SEQ ID NO:2-66.

In another aspect, the present invention provides polypeptidescomprising an amino acid sequence according to SEQ ID NO:1, wherein thepolypeptide comprises at least one amino acid change from SEQ ID NO:33.In another aspect, the present invention provides a polypeptidecomprising an amino acid sequence according to SEQ ID NO:35, wherein thepolypeptide comprises at least one amino acid change from SEQ ID NO:67.In various embodiments, the polypeptide, comprises an amino acidsequence according to any one of SEQ ID NO:2-66.

In a further aspect the present invention provides nucleic acidsencoding the polypeptide of any aspect or embodiment of the invention.In another aspect, the invention provides nucleic acid expressionvectors comprising the isolated nucleic acids of the invention. In afurther embodiment, the invention provides recombinant host cellscomprising the nucleic acid expression vectors of the invention. Inanother aspect, the invention provides pharmaceutical compositions,comprising the polypeptides, the nucleic acids, the nucleic acidexpression vectors and/or the recombinant host cells of the invention,and a pharmaceutically acceptable carrier

In another aspect, the invention provides methods for treating celiacsprue, comprising administering to an individual with celiac sprue apolypeptide of pharmaceutical composition according to any embodiment ofthe invention, or a polypeptide comprising an amino acid selected fromthe group consisting of SEQ ID NO:33 or SEQ ID NO:67.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic depicting the role of enzyme therapeutics in thetreatment of Celiac disease. Gluten is comprised of many glycoproteinsincluding α-gliadin. Partial proteolysis of α-gliadin (SEQ ID NO:71)results in protease-resistance peptides enriched in a PQ dipeptide motifthat can lead to inflammation and disease. The addition of an oralenzyme therapeutic that is funcetional in the stomach and capable ofspecifically degrading the immunogenic peptides could potentially act asa therapeutic for this disease.

FIG. 2. Computational models of the peptide binding sites for KumaWT andKumaMax. A) KumaWT in complex with a PR dipeptide motif. B) KumaMax incomplex with the designed PQ dipeptide motif. Computationally designedresides in the active site are labeled and highlighted in sticks. Themodeled peptdes were based on a bound form of Kumamolisin-AS (PDB ID:IT1E) and final structures were generated using the Rosetta MolecularModeling Suite. Images were generated using PyMol v1.5.

FIG. 3. Protein stability after incubation with pepsin or trypsin.Stability was measured by quantifying the relative remaining fraction ofintact protein as observed on an SDS-PAGE gel after 30 minutes ofincubation in the presence or absence of pepsin or trypsin at the pHindicated. Each protein was measured in triplicate and the error barsrepresent the standard deviation. Quantification was performed inImageJ™.

FIG. 4. An immunogenic α9-gliadin peptide is degraded by KumaMax. A)Reaction chromatograms measuring the abundance of the M+H ion of theparent α9-gliadin peptide after 50 minutes of incubation with no enzymenSC PEP, or KumaMax™. B) The fraction of α9-gliadin peptide remaining inthe presence of KumaMax™ as a function of incubation time at pH 4. Thedata was fit using a standard exponential decay function. The R² valuewas greater than 0.9.

DETAILED DESCRIPTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniqaues utilized my be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989.Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Val. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purrification” in Methodsin Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc. ); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990,Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney, 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferent unless the contest clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, ammo acid residues are abbreviated as follows; alanine(Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg;R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q),glycine (Gly, G), histidine (His; H), isoleucine (Ile; I), leucine (Leu;L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F),proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp;W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides polypeptidescomprising an amino acid sequence at least 75% identical to an aminoacid sequence according to SEQ ID NO:33, wherein

(a) the polypeptide degrades a PQPQLP (SEQ ID NO:34) peptide at pH 4;

(b) residue 278 is Ser, residue 78 is Glu, and residue 82 is Asp; and

(c) the polypeptide comprises an amino acid change from SEQ ID NO:67 atone or more resides selected from the group consisting of 73, 102, 103,104, 130, 168, 169, 172, and 179.

As disclosed in the examples that follow, polypeptides according to thisaspect of the invention can be used, for example, in treating celiacsprue. The polypeptides are modified versions of either the processedversion of Kumamolisin-As (SEQ ID No:67) or the preprocessed version ofKumamolisin-As (SEQ ID NO:33), which is known as a member of thesedolisin family of serine-carboxyl peptidases, and utilizes the keycatalytic triad Ser²⁷⁸-Glu⁷⁸-Asp⁸² in its processed form to hydrolyzeits substrate (Ser⁴⁶⁷-Glu²⁶⁷-Asp²⁷¹ in the preprocessed form) Itsmaxiamal activity is at pH ˜4.0. While the native substrate forKumamolisin-As is unknown, it has been previously shown to degradecollagen under acidic conditions (4). In addition, this enzyme has beenshown to be thermostable, with an ideal temperature at 60° C. but stillshowing significant activity at 37° C.

The invention of the present invention have unexpectedly discovered thatKumamolisin-As is capable of degrading proline (P)- and glutamine(Q)-rich components of gluten known as ‘gliadins’ believed responsiblefor the bulk of the immune response in most celiac sprue patients. Themolypeptides of the invention show improved protease activity at pH 4against the oligopeptide PQPQLP (a substrate representative of gliadin)compared to wild type Kumamolisin-As.

The polypeptides of this aspect of the invention degrade a PQPQLP (SEQID NO:34) peptide at pH 4. Such degradation occurs under the conditionsdisclosed in the examples that follow.

The polypeptides of this aspect comprise one or more amino acid changesfrom SEQ ID NO: 67 (wild type processed Kumamolisin-As) at one or moreresides selected from the group consisting of residues 73, 102, 103,104, 130, 165, 168, 169, 172, and 179 (numbering based on the wild typeprocessed Kumamolisin-As amino acid sequence). In non-limitingembodiments, the one or more changes relative to the wild type processedKumamolisin-As amino acid sequence (SEQ ID NO:67) are selected from thegroup consisting of:

WT Residue# AA change S73 K, G N102 D T103 S D104 A, T, N G130 S S165 NT168 A D169 N, G Q172 D D179 S, H

In various further non-limiting embodiments, the one or more changesrelative to the wild type processed Kumamolisin-As amino acid sequenceinclude at least N102D. In another embodiment the one or more changesrelative to the wild type Kumamolisin-As amino acid sequence include atleast N102D and D169N or D169G. In another embodiment the one or morechanges relative to the wild type Kumamolisin-As amino acid sequenceinclude at least N102D, D169G, and D179H. In another embodiment the oneor more changes relative to the wild type Kumamolisin-As amino acidsequence include at least S73K, D104T, N102D, G130S, D169G, and D179H.

As used herein, “at least 75% identical” means that the polypeptidediffers in its full length amino acid sequence by less 25% or less(including any amino acid substitutions, deletions, additions, orinsertions) from the polypeptide defined by SEQ ID NO:35.

In various preferred embodiment, the polypeptides comprise or consist ofan amino acid sequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence according to SEQ IDNO:35. In a further embodiment of the polypeptides comprise or consistof an amino acid sequence according to SEQ ID NO:35.

In various further embodiments, the polypeptides comprise or consist ofan amino acid sequence at least 75% identical to any one of SEQ IDNOS:36-66. The polypeptides represented by these SEQ ID NOS are specificexamples of polypeptides with improved protease activity of pH 4 againstthe oligopeptide PQPQLP (SEQ ID NO:34) (a substrate representative ofgliadin) compared to wild type Kumamolisin-As. In various preferredembodiment, the polypeptide s comprise or consist of an amino acidsequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence according to any one of SEQ IDNOS:36-66. In a further embodiment the polypeptides comprise or consistof an amino acid sequence according to any one of SEQ ID NOS:36-66.

In a preferred embodiment of this first aspect, the polypeptidescomprising an amino acid sequence at least 75% identical to an aminoacid sequence according to SEQ ID NO:1 (based on variants of thepreprocessed version of Kumamolisin-As), wherein

(a) the polypeptide degrades a PQPQLP (SEQ ID NO:34) peptide at pH 4;

(b) residue 467 is Ser, residue 267 is Glu, and residue 271 is Asp; and

(c) the polypeptide comprises an amino acid change from SEQ ID NO:33 atone or more resides selected from the group consisting of 119, 262, 291,292, 293, 319, 354, 357, 358, 361, and 368.

The polypeptides of this embodiment comprise one or more amino acidchanges from SEQ ID NO:33 (wild type pre-processed Kumamolisin-As) atone or more residues selected from the group consisting of residues 119,262 , 291, 292, 293, 319, 354, 357, 358, 361, and 368 (numbering basedon the wild type pre-processed Kumamolisin-As amino acid sequence). Innon-limiting embodiments, the one or more changes relative to the wildtype Kumamolisin-As amino acid sequence are selected from the groupconsisting of:

WT Residue# AA change V119 D S262 K, G N291 D T292 S D293 A, T, N G319 SS354 N T357 A D358 N, G Q361 D D368 S, H

In various further non-limiting embodiments, the one or more changesrelative to the wild type Kumamolisin-As amino acid sequence include atleast N291D. In another embodiment the one or more changes relative tothe wild type Kumamolisin-As amino acid sequence include at least N291Dand 358N or 358G. In another embodiment the one or more changes relativeto the wild type Kumamolisin-As amino acid sequence include at leastN291D, 358G, and 368H. In another embodiment the one or more changesrelative to the wild type Kumamolisin-As amino acid sequence include atleast V119D, S262K, D293T, N291D, G319S, D358G, and D369H.

As used herein, “at least 75% identical” means that the polypeptidediffers in its full length amino acid sequence by less 25% or less(including any amino acid substitutions, deletions, additions, orinsertions) from the polypeptide defined by SEQ ID NO:1.

In various preferred embodiment, the polypeptides comprise or consist ofan amino acid sequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 790%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence according to SEQ IDNO:1. In a further embodiment the polypeptides comprise or consist of anamino acid sequence according to SEQ ID NO:1.

In various further embodiments, the polypeptides comprise or consist ofan amino acid sequence at least 75% identical to any one of SEQ IDNOS:2-32. The polypeptides represented by these SEQ ID NOS are specificexamples of polypeptides with improved protease activity at pH 4 againstthe oligopeptide PQPQLP (SEQ ID NO:34) (a substrate representative ofgliadin) compared to wild type Kumamolisin-As. In various preferredembodiment, the polypeptides comprise or consist of an amino acidsequence at least 76%, 77%, 78%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence according to any one of SEQ IDNOS:2-32. In a further embodiment the polypeptides comprise or consistof an amino acid sequence according to any one of SEQ ID NOS:2-32.

In a second aspect, the present invention provides polypeptides,comprising or consisting of an amino acid sequence according to SEQ IDNO:35, wherein the polypeptide comprises at least one amino acid changefrom SEQ ID NO:67. As disclosed in the examples that follow,polypeptides according to this aspect of the invention can be used, forexample, in treating celiac sprue. The polypeptides are modifiedversions of processed Kumamolisin-As (SEQ ID NO:67), that show improvedprotease activity at pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34)(a substrate representative of gliadin) compared to wild typeKumamolisin-As. In one embodiment, the polypeptides comprise or consistof an amino acid sequence according to SEQ ID NO:36. Polypeptidesaccording to SEQ ID NO:36 have a N102D mutation relative to wild-typeprocessed Kumamolisin-As. As shown in the examples that follow,polypeptides containing this mutation have at least 10-fold improvedprotease activity at pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34)compared to wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptides compriseof consist of an amino acid sequence according to SEQ ID NO:37.Polypeptides according to SEQ ID NO:37 have a N102D mutation and a D169Nor D169G mutation to wild-type processed Kumamolisin-As. As shown in theexample that follow, polypeptides containing the mutation have at least20-fold improved protease activity at pH 4 against the oligopeptidePQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptides compriseor consist of an amino acid sequence according to SEQ ID NO:38.Polypeptides according to SEQ ID NO:38 have a N102D mutation a D169G,and a D179H mutation relative to wild-type processed Kumamolisin-As. Asshown in the examples that follow, polypeptides containing this mutationhave at least 50-fold improved protease activity at pH 4against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.

In a further embodiment of this second aspect, the polypeptides compriseor consist of an amino acid sequence according to any one of SEQ IDNOS:39_66. Polypeptides according to these embodiments have all beendemonstraated to show improved protease activity at pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.In a preferred embodiment, the polypeptide comprises or consists of anamino acid sequence according to SEQ ID NO:66.

In a preferred embodiment of this second aspect, the present inventionprovides polypeptides comprising or consisting of an amino acid sequenceaccording to SEQ ID NO:1, wherein the polypeptide comprises at least oneamino acid change from SEQ ID NO:33. As disclosed in the examples thatfollow, polypeptides according to this aspect of the invention can beused, for example, in treating celiac sprue. The polypeptides aremodified versions of preprocessed Kumamolisin-As (SEQ ID NO:33), thatshow improved protease activity at pH 4 against the oligopeptidePQPQLP(SEQ ID NO:34) (a substrate representative of gliadin) compared towild type Kumamolisin-As. In one embodiment, the polypeptides compriseor consist of an amino acid sequence according to SEQ ID NO:2.Polypeptides according to SEQ ID NO:2 have a N291D mutation relative topreprocessed wild-type Kumamolisin-As, As shown in the examples thatfollow, polypeptides containing this mutation have at least 10-foldimproved protease activity at pH 4 against the oligopeptide PQPQLP (SEQID NO:34) compared to wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptides compriseor consist of an amino acid sequence according to SEQ ID NO:3.Polypeptides according SEQ ID NO:3 have a N291 D mutation and a D358N orD358G mutation relative to preprocessed wild-type Kumamolisin-As. Asshown in the examples that follow, polypeptides containing this mutationhave at least 20-fold improved protease activity at pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptides compriseor consist of an amino acid sequence according to SEQ ID NO:4.Polypeptides according to SEQ ID NO:4 have a N292D mutation, a D358G,and a D368H mutation relative to preprocessed wild-type Kumamolisin-As.As shown in the examples that follow, polypeptides containing thismutation have at least 50-fold improved protease activity at pH 4against the oligopeptide PQPQLP (SEQ ID NO:34) compared to wild typeKumamolism-As.

In a further embodiment of this second aspect, the polypeptides compriseor consist of an amino acid sequence according to any one of SEQ IDNOS:5-32. Polypeptides according to these embodiments have all beendemonstrated to show improved protease activity at pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.In a preferred embodiment, the polypeptide comprises or consists of anamino acid sequence according to SEQ ID NO:32; this polypeptide is shownin the examples that follow to possess the most-potent protease activityat pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34) of any of thepolypeptides tested.

As used throughtout the present application, the term “Polypeptide” isused in its broadest sense to refer to a sequence of subunit aminoacids, whether naturally occurring or of synthetic origin. Thepolypeptides of the invention may comprise L-amino acids. D-amino acids(which are resistant to L-amino acid-specific proteases in vivo), or acombination of D- and L-amino acids. The polypeptides described hereinmay be chemically synthesized or recombinantly expressed. Thepolypeptides may be linked to other compounds to promote an increasedhalf life in vivo, such as by PEGylation, HESylation, PASlation, orglycosylation. Such linkage can be covalent or non-covalent as isunderstood by those of skill in the art. The polypeptides may be linkedto any other suitable linkers, including but not limited to any linkersthat can be used for purification or detection (such as FLAG or Histags).

In a third aspect, the present invention provides isolated nucleic acidsencoding the polypeptide of any aspect or embodiment of the invention.The isolated nuleic acid sequence may comprise RNA or DNA. As usedherein, “isolated nucleic acids” are those that have been removed fromtheir normal surrounding nucleic acid sequences in the genome or in cDNAsequences. Such isolated nucleic acid sequences may comprise additionalsequences useful for promoting expression and/or purification of theencoded protein, including but not limited to poly A sequences, modifiedKozak sequences, and sequences encoding epitope tags, export signals,and secretory signals, nuclear localization signals, and plasma membranelocalization signals. It will be apparent to those of skill in the art,based on the teaching herein, what nucleic acid sequences will encodethe polypeptides of the invention.

In a fourth aspect, the present invention provides nucleic acidexpression vectors comprising the isolated nucleic acid of anyembodiment of the invention operatively linked to a suitable controlsequence. “Recombinant expression vector” includes vectors thatoperatively link a nucleic acid coding region or gene to any controlsequences capable of effecting expression of the gene product. “Controlsequences” operable linked to the nucleic acid sequences of theinvention are nucleic acid sequences capable of effecting the expressionof the nucleic acid molecules. The control sequences need not becontiguous with the nucleic acid sequences, so long as they function todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the nucleic acid sequences and the promoter sequence canstill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, termination signals, and ribosome binding sites. Suchexpression vectors ca be of any type known in the art, including but notlimited plasmid and viral-based expression vectors. The control sequenceused to drive expression of the disclosed nucleic acid sequences in amammalian system may be constitutive (driven by any of a variety ofpromoters, including but not limited to, CMV, SV40RSV, actin, EF) orinducible (driven by any of a number of inducible promoters including,but not limited to, tetracycline, ecdysone, steroid-responsive). Theconstruction of expression vectors for us in transfecting prokaryoticcells is also well known in the art, and thus can be accomplised viastandard techniques. (See, for example, Sambrook, Fritsch, and Maniatis,in: Molecular Cloning, A laboratory Manual, Cold Spring HarborLaboratory Press, 1989; Gene Transfer and Expression Protocols, pp.109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), andthe Ambion 1998 Catalog (Ambion, Austin, Tex.). The expression vectormust be replicable in the host organisms either as an episome or byintegration into host chromosomal DNA. In a preferred embodiment, theexpression vector comprises a plasmid. However, the invention isintended to include other expression vectors that serve equivalenfunctions, such as viral vectors.

In a fifth aspect, the present invention provides recombinant host cellscomprising the nucleic acid expression vectors of the invention. Thehost cells can be either prokaryotic or eukaryotic. The cells can betransiently or stably transfected or transduced. Such transfection andtransduction of expression vectors into prokaryotic and eukaryotic cellscan be accomplished via any technique known in the art, including butnot limited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome midiated-, DEAE dextranmediated-, polycationic medicated-, or viral mediated transfection.(See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, etal., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells:A Manual of Basic Technique, 2^(nd) Ed. (R. I. Freshney, 1987, Liss,Inc. New York, N.Y.). A method of producing a polypeptide according tothe invention is an additional part of the invention. The methodcomprises the steps of (a) culturing a host according to this aspect ofthe invention under conditions conducive to the expression of thepolypeptide, and (b) optionally, recovering the expressed polypeptide.The expressed polypeptide can be recovered from the cell free extract,cell pellet, or recovered from the culture medium. Methods to purifyrecombinantly expressed polypeptides are well known to the man skilledin the art.

In a sixth aspect, the present invention provides pharmaceuticalcompositions, compring the polypeptide, nucleic acid, nucleic acidexpression vecto, and/or the recombinant host cell of any aspect orembodiment of the invention, and a pharmaceutically acceptable carrier.The pharmaceutical compositions of the invention can be used, forexample, in the methods of the invention described below. Thepharmaceutical composition may comprise addition to the polypeptides,nucleic acids, etc. of the invention (a) a lyoprotectant; (b) asurfactant, (c) a bulking agent; (d) a tonicity adjusting agent; (e) astabilizer; (f) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is aTris buffer, a histidine buffer, a phosphate buffer, a citrate buffer oran acetate buffer. The pharmaceutical composition may also include alyoprotectant, e.g. sucrose, sorbitol of trehalose. In certainembodiments, the pharmaceutical composition includes a preservative e.g.benzalkonium chloride, benzethonium, chlorohexidine, phenol, n-cresol,benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonocleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The polypeptides, nucleic acids, etc. of the invention may be the soleactive agent in the pharmaceutical composition, or the composition mayfurther comprise one or more other active agents suitable for anintended use.

The pharmaceutical compositions described herein generally comprise acombination of a compound described herein and a pharmaceuticallyacceptable carrier, diluents, or excipient. Such compositions aresubstantially free of non-pharmaceutically acceptable components, i.e.,contain amounts of non-pharmaceutically acceptable components lower thanpermitted by US regulatory requirements at the time of filing thisapplication. In some embodiments of this aspect, if the compound isdissolved or suspended in water, the composition further optionallycomprises an additional pharmaceutically acceptable carrier, diluent, ofexcipient. In other embodiments, the pharmaceutical compositionsdescribed herein are solid pharmaceutical compositions (e.g., tablet,capsules, etc.).

These compositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by any suitable route. In apreferred embodiment, the pharmaceutical compositions and formulationsare designed for oral administration. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

The pharmaceutical compositions can be in any suitable form, includingbut not limited to tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules, sterileinjectable solutions, and sterile packaged powders.

In a seventh aspect, the present invention provides methods for treatingceliac sprue, comprising administering to an individual with celiacsprue an amount effective to treat the celiac sprue of one or morepolypeptides selected from the group consisting of the polypeptides ofthe first or second aspects of the invention, SEQ ID NO:33, and SEQ IDNO:67.

The inventors of the present invention have unexpectedly discovered thatKumamolisin-As is capable of degrading proline (P)- and glutamine(Q)-rich components of gluten known as ‘gliadins’ believed responsiblefor the bulk of the immune response in most celiac sprue patients. Thepolypeptides of the invention show improved protease activity at pH 4against the oligopeptide PQPQLP (SEQ ID NO:34) (a substraterepresentative of gliadin) compared to wild type Kumamolisin-As.

In one embodiment, the one or more polypeptides comprise an amino acidsequence at least 75% identical to an amino acid sequence according toSEQ ID NO:35, wherein

(a) the polypeptide degrades a PQPQLP (SEQ ID NO:34) peptide at pH 4;

(b) residue 278 is Ser, residue 78 Glu, and residue 82 is Asp.

In further embodiments, the one or more polypeptides comprise one ormore amino acid changes from SEQ ID NO:67 (wild type processedKumamolisin-As) at one or more residues selected from the groupconsisting of residues 73, 102, 103, 104, 130, 165, 168, 169, 172, and179 (numbering based on the wild type processed Kumamolisin-As aminoacid sequence). In non-limiting embodiments, the one or more changesrelative to the wild type processed Kumamolisin-As amino acid sequence(SEQ ID NO:67) are selected from the group consisting of:

WT Residue# AA change S73 K, G N102 D T103 S D104 A, T, N G130 S S165 NT168 A D169 N, G Q172 D D179 S, H

In various further non-limiting embodiments, the one or more changesrelative to the wild type process Kumamolisin-As amino acid sequenceinclude at least N102D. In another embodiment the one or more changesrelative to the wild type Kumamolisin-As amino acid sequence include atleast N102D and D169N or D169G. In another embodiment the one or morechanges relative to the wild type Kumamolisin-As amino acid sequenceinclude at least N102D and D169N or D169G. In another embodiment the oneor more changes relative to the wild type Kumamolisin-As amino acidsequence include at least S73K, D104T, N102D, G130S, D169G, and D179H.

In various preferred embodiment, the one or more polypeptides compriseor consist of an amino acid sequence at least 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 790%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence accordingto SEQ ID NO:35. In a further embodiment the polypeptides comprise orconsist of an amino acid sequence according to SEQ ID NO:35.

In various further embodiments, the polypeptides comprise or consist ofan amino acid sequence at least 75% identical to any one of SEQ IDNOS:36-66. The polypeptides represented by these SEQ ID NOS are specificexamples of polypeptides with improved protease activity at pH 4 againstthe oligopeptide PQPQLP (SEQ ID NO:34) (a substrate representative ofgliadin) compared to wild type Kumamolisin-As. In various preferredembodiment, the polypeptides comprise or consist of an amino acidsequence at least 76%, 77%, 78%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence according to any one of SEQ IDNOS:36-66. In a further embodiment the polypeptides comprise or consistof an amino acid sequence according to any one of SEQ ID NOS:36-66.

In a preferred embodiment, the polypeptides for use in the methods ofthis aspect of the invention comprise an amino acid according to SEQ IDNO:33 or a polypeptide comprising one or more amino acid changes fromSEQ ID NO:33 (wild type preprocessed Kumamolisin-As) at one or moreresidues selected from the group consisting of residues 119, 262, 291,292, 293, 319, 354, 357, 358, 361, and 368 (numbering based on the wildtype Kumamolisin-As amino acid sequence). In non-limiting embodiments,the one or more changes relative to the wild type Kumamolisin-As aminoacid sequence are selected from the group consisting of:

WT Residue# AA change V119 D S262 K, G N291 D T292 S D293 A, T, N G319 SS354 K T357 A D358 N, G Q361 D D368 S, H

In various further non-limiting embodiments, the one or more changesrelative to the wild type processed Kumamolisin-As amino acid sequenceinclude at least N291D. In another embodiment the one or more changesrelative to the wild type Kumamolisin-As amino acid sequence include atleast N291D and 358N or 358G. In another embodiment the one or morechanges relative to the wild type Kumamolisin-As amino acid sequenceinclude at least N291D, 358G, and 368H. In another embodiment the one ormore changes relative to the wild type Kumamolisin-As amino acidsequence include at least V119D, S262K, D293T, N291D, G319S, D358G, andD368H.

In various preferred embodiment, the polypeptides for use in the methodsof this aspect of the invention comprise or consist of an amino acidsequence at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence according to SEQ ID NO:1. In afurther embodiment of the polypeptides comprise or consist of an aminoacid sequence according to SEQ ID NO:1.

In various further embodiments, the polypeptides for use in the methodsof this aspect of the invention comprise or consist of an amino acidsequence at least 75% identical to any one of SEQ ID NOS:2-32. Thepolypeptides represented by these SEQ ID NOS are specific examples ofpolypeptides with improved protease activity of pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) (a substrate representative ofgliadin) compared to wild type Kumamolisin-As. In various preferredembodiment, the polypeptides for use in the methods of this aspect ofthe invention comprise or consist of an amino acid sequence at least76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to anamino acid sequence according to any one of SEQ ID NOS:2-32. In afurther embodiment the polypeptides comprise or consist of an amino acidsequence according to any one of SEQ ID NOS:2-32.

In an eighth aspect, the present invention provides methods for treatingceliac sprue, comprising administering to an individual with celiacsprue a polypeptide comprising an amount effective of amino acidsequence according to any one of SEQ ID NOS:1-67 to treat the celiacsprue.

In one embodiment, the polypeptides administered comprise or consist ofan amino acid sequence according to SEQ ID NO:2. Polypeptides accordingto SEQ ID NO:2 have a N291D mutation relative to wild-typeKumamolisin-As. As shown in the examples that follow, polypeptidescontaining this mutation have at least 10-fold improved proteaseactivity at pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34) comparedto wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptidesadministered comprise or consist of an amino acid sequence according toSEQ ID NO:3. Polypeptides according to SEQ ID NO:3 have a N291D mutationand a D358N or D358G mutation relative to wild-type Kumamolisin-As. Asshown in the examples that follow polypeptides containing this mutationhave at least 20-fold improved protease activity at pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.

In another embodiment of this second aspect, the polypeptidesadministered comprise or consist of an amino acid sequence according toSEQ ID NO:4. Polypeptides according to SEQ ID NO:4 have a N291D mutationand a D358G, and a D368H mutation relative to wild-type Kumamolisin-As.As shown in the examples that follow polypeptides containing thismutation have at least 50-fold improved protease activity at pH 4against the oligopeptide PQPQLP (SEQ ID NO:34) compared to wild typeKumamolisin-As.

In a further embodiment of this second aspect, the polypeptidesadministered comprise or consist of an amino acid sequence according toany one of SEQ ID NOS:5-32. Polypeptides according to these embodimentshave all been demonstrated to show improved protease activity of pH 4against the oligopeptide PQPQLP (SEQ ID NO: 34) compared to wild typeKumamolisin-As. In a preferred embodiment, the polypeptide administeredcomprises or consists of an amino acid sequence according to SEQ IDNO:32; this polypeptide is shown in the examples that follow to possessthe most potent protease activity at pH 4 against the oligopeptidePQPQLP (SEQ ID NO:34) of any of the polypeptides tested.

In another embodiment, the one or more polypeptides comprise an aminoacid sequence according to SEQ ID NO:36. Polypeptides according to SEQID NO:36 have a N102D mutation relative to wild-type processedKumamolisin-As. As shown in the examples that follow, polypeptidescontaining this mutation have at least 10-fold improved proteaseactivity at pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34) comparedto wild type Kumamolisin-As. In another embodiment, the one or morepolypeptides comprise an amino acid sequence according to SEQ ID NO:37.Polypeptides according to SEQ ID NO:37 have a N102D mutation and a D169Nor D169G mutation relative to wild-type processed Kumamolisin-As. Asshown in the examples that follow, polypeptides containing this mutationhave at least 20-fold improved protease activity at pH 4 against theoligopeptide PQPQLP (SEQ ID NO:34) compared to wild type Kumamolisin-As.In another embodiment, the one or more polypeptides comprise an aminoacid sequence according to SEQ ID NO:38. Polypeptides according to SEQID NO:38 have a N102D mutation, a D169G, and a D179H mutation relativeto wild-type processed Kumamolisin-As. As shown in the examples thatfollow, polypeptides containing this mutation have at least 50-foldimproved protease activity at pH 4 against the oligopeptide PQPQLP (SEQID NO:34) compared to wild type Kumamolisin-As. In further embodiments,the one or more polypeptides comprise an amino acid sequence accordingto any one of SEQ ID NOS:39-66. Polypeptides according to theseembodiments have all been demonstrated to show improved proteaseactivity at pH 4 against the oligopeptide PQPQLP (SEQ ID NO:34) comparedto wild type Kumamolisin-As. In a preferred embodiment, the polypeptidecomprises or consists of an amino acid sequence according to SEQ IDNO:66.

Celiac sprue (also known as celiac disease or gluten intolerance) is ahighly prevalent disease in which dietary proteins found in wheat,barley, and rye products known as ‘glutens’ evoke an immune response inthe small intestine of genetically predisposed individuals. Theresulting inflammation can lead to the degradation of the villi of thesmall intestine, impeding the absorption of nutrients. Symptoms canappear in early childhood or later in life, and range widely inseverity, from diarrhea, fatigue, weight loss, abdominal pain, bloating,excessive gas, indigestion, constipation, abdominal distension,nausea/vomiting, anemia, bruising easily, depression, anxiety, growthdelay in children, hair loss, dermatitis, missed menstrual periods,mouth ulcers, muscle cramps, joint pain, nosebleeds, seizures, tinglingor numbness in hands or feet, delayed puberty, defects in tooth enamel,and neurological symptoms such as ataxia or paresthesia. There arecurrently no effective therapies for this lifelong disease except thetotal elimination of glutens from the diet. Although celiac sprueremains largely underdiagnosed, its' prevalence in the US and Europe isestimated at 0.5-1.0% of the population.

As used herein, “treating celic sprue” means accomplishing one or moreof the following: (a) reducing the severity of celiac sprue; (b)limiting or preventing development of symptoms, characteristic of celiacsprue; (c) inhibiting worsening of symptoms characteristic of celiacsprue; (d) limiting or preventing recurrence of celiac sprue in patientsthat have previously had the disorder; (e) limiting or preventingrecurrence of symptoms in patients that were previously symptomatic forceliac sprue; and (f) limiting development of celiac sprue in a subjectat risk of developing celiac sprue, or not yet showing the clinicaleffects of celiac sprue.

The individual to be treated according to the methods of the inventionmay be any individual suffering from celiac sprue, including humansubjects. The individual may be one already suffering from symptoms orone who is asymptomatic.

As used herein, an “amount effective” referred to an amount of thepolypeptide that is effective fore treating celiac sprue. Thepolypeptides are typically formulated as a pharmaceutical composition,such as those disclosed above, and can be administered via any suitableroute, including orally, parentally, by inhalation spray, or topicallyin dosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles. In a preferred embodiment,the pharmaceutical compositions and formulations are orallyadministered, such as by tablets, pills, lozenges, elixirs, suspensions,emulsions, solutions, or syrups.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). A suitable dosage rangemay, for instance, be 0.1 ug/kg-100 mg/kg body weight; alternatively, itmay be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10mg/kg body weight. The polypeptides can be delivered in a single bolus,or may be administered more than once (e.g., 2, 3, 4, 5, or more times)as determined by an attending physician.

EXAMPLES

Celiac disease is an autoimmune disorder that afflicts approximately 1%of the population^(1.2). This disease is characterized by aninflammatory reaction to gluten, the major protein in wheat flour, andto related proteins in barley and rye². Gluten is composed of aheterogeneous mixture of the glycoproteins gliadin and glutenin³. Uponingestion, α-gliadin is partially degraded by gastric and intestinalproteases to oligopeptides, which are resistant to further proteolysisdue to their unusually high proline and glutamine content² (FIG. 1).Immunogenic oligopeptides that result from incomplete proteolysis areenriched in the PQ motif^(3.5) (FIG. 1), which stimulate inflammationand injury in the intestine of people with Celiac disease. Currently theonly treatment for this disease is complete elimination of gluten fromthe diet, which is difficult to attain due to the ubiquity of thisprotein in modern food products⁶.

Oral enzyme therapy (OFT) in which orally administered proteases areemployed to hydrolyze immunogenic peptides before they are capable oftriggering inflammation is currently being explored as a treatment forgluten intolerance. For this purpose, several different proteases havebeen considered due to their specificity for cleavage after eitherproline or glutamine residues^(6.2.8). However, these enzymes oftendemonstrate characteristics that hinder their use in OET for glutendegradation. Most of these peptidases exhibit optimal catalytic activityat neutral pH, however, the pH of the human stomach ranges from 2 to 4.These enzymes are therefore most active when they reach the pH-neutralsmall intestine, which is too late for effective prevention of Celiacdisease as this is the site where gluten-derived pathologydevelops^(2.16). Additionally, several of these enzymes demonstrateinstability in the low pH of the human stomach, are susceptible toproteolysis by digestive proteases, or require extensive refoldingprocedures during their purification^(2.11), which are allcharacteristics that hamper efforts for clinical use.

The ideal protease for the application of OET in the treatment of glutenintolerance would combine the following traits: optimal activity at lowpH, easy purification, stability under the conditions of the humanstomach, and high specificity for amino acid motifs found ingluten-derived immunogenic oligopeptides. Here we report the engineeringof an endopeptidase that demonstrates these traits. We identified aprotease that is highly active in acidic conditions, Kumamolisin-As(Kuma WT) from the acidophilic bacterium Alicyclobacillus sendaiensis,and used computational modeling tools to engineer it toward the desiredoligopeptide specificity. The computationally designed enzyme,designated KumaMax™, exhibited over 100fold increased proteolyticactivity and an 800-fold switch in substrate specificity for thetargeted PQ motif compared to wild-type KumaWT. In addition, KumaMax™demonstrates resistance to common gastric proteases and is produced athigh yields in E. coli without the need for refolding. Thus, thisprotease and others reported herein represent promising therapeuticcandidates for Celiac disease.

RESULTS Selection and Computational Design of an α-Gliadin Endopeptidase

In order to engineer a novel protease that can degrade gluten peptidesunder gastric conditions, we first focused on identifying an appropriateprotease as a starting point for our engineering efforts. Ideally, thetemplate protease would combine stability and activity at low pH withdemonstrated specificity for a dipeptide amino acid motif. We identifiedthe enzyme Kumamolisin-As (KumaWT) as a template, since this proteasenaturally has an optimal activity over the pH range of 2-4¹², whichmatches the approximate pH ranges in the human stomach before and aftera meal is ingested (pH 2 and 4, respectively)¹². KumaWT alsodemonstrates high stability and activity at the physiologically relevanttemperature 37° C.¹⁶. In addition, the purification of this enzyme isstraightforward and yields significant quantities using standardrecombinant protein production methods in E. coli ¹³, an importantproperty both for screening mutant libraries and for its ultimategeneration in large batches for use in OET. Finally, KumaWT naturallyrecognizes a specific dipeptide motif as opposed to single amino acidspecificity¹⁵. This is an important property for an oral proteasetherapeutic meant to be takes during digestion, since dipeptidespecificity should result in reduced competitive inhibition by otherfood-derived peptides in the stomach.

An effective OET for Celiac disease would likely demonstrate specificitytor Proline-Glutamine (PQ), due to the frequent occurrence of thisdipeptide in immunogenic gluten-derived oligopeptides (FIG. 1). KumaWThas a strong specificity for proline at the P2 position of its peptidesubstrate, matching one of the amino acid residues of interest for thedegradation of immunogenic α-gliadin peptides. In the P1 site, KumaWThas been established to prefer the-positively charged amino acidsarginine or lysine¹³. Despite this preference, KumaWT is also capable ofrecognizing glutamine-at the P1 position, albeit at a significantlydecreased level compared to its recognition of arginine or lysine¹⁴.This slight innate proclivity to recognize glutamine at the P1 positionsuggests that KumaWT may be amenable to re-engineering to preferglutamine at this position. At the P1′ site, KumaWT demonstrates broadspecificity, which is desirable since the residue in the position afterthe PQ motif varies among the different immunogenic peptides, asdepicted in FIG. 1.

Given these characteristics of KumaWT, our primary goal was tocomputationally redesign the S1 binding pocket of KumaWT such that itwould prefer a PQ dipeptide motif over the native PR or PK substrates.Using the Rosetta Molecular Modeling Suite, we modeled the PR dipeptidein the S1 binding pocket of KumaWT using this enzyme's solved crystalstructure (PDB ID: 1T1E). This revealed that two negatively-chargedamino acids, D358 and D368, likely facilitate binding of the positivelycharged amino acids in the P1 position (FIG. 2A). The native specificityfor proline at P2 appears to be derived in large part from a hydrophobicinteraction of this amino acid residue with the aromatic ring of W318 inthe S2 pocket of the enzyme. As specificity of the P1 position forproline is desired in our enzyme variant, we maintained this nativetryptophan during the design of the S1 pocket.

To redesign the KumaWT substrate specificity of the S1 pocket to preferglutamine at the P1 position, we generated theoretical mutations in theKumaWT binding pocket using the Foldit interface to the RosettaMolecular Modeling Suite. A tetrapeptide that represents a commonimmunogenic motif found throughout α-gliadin, PQLP (SEQ ID NO:68), wasmodeled into the P2 to P2′ active site positions. This structure alreadycontained a polypeptide bound in the active site, so the residues ofthis polypeptide were mutated using Rosetta to the PQLP (SEQ ID NO:68)tetrapeptide motif. A total of 75 residues within an 8Å sphere of thetetrapeptide were randomized to any of the 20 naturally occurring aminoacids in order to find mutations that would favor binding of glutaminein the S1 pocket. These mutations were accepted if the overall energy ofthe new enzyme-PQLP substrate complex was either reduced relative to thenative substrate, or was not increased y more than 5 Rosetta energyunits. To accommodate the smaller, neutral amino acid glutamine, wefocused our computational efforts on 1) removing the negative charge ofthe S1 pocket during the design process, 2) filling in open space thatresulted from the replacement of the large amino acid arginine withglutamine, and 3) providing hydrogen bonds to the amid functional groupof the glutamine. This computational modeling yielded 107 novel designscontaining from 1 to 7 simultaneous mutations. These designed proteinswere then constructed and their catalytic activity against a PQLP (SEQID NO:68) peptide was assessed.

In order to test the activity of each of these designed proteasesagainst the PQLP (SEQ ID NO:68) motif, the desired mutations wereincorporated into the native nucleotide sequence using site directedmutagenesis, and mutant enzyme variants were produced in E. coli BL21(DE3) cells. These enzyme variants were then screened for enzymaticactivity in clarified whole cell lysates at pH 4 using the fluorescentlyquenched α-gliadin hexapeptide analogue QXL520-PQPQLP-K(5-FAM)-NH2 (FQ)(SEQ ID NO:69) as a substrate. Of the 107 enzyme variants tested in thisassay, 13% resulted in a loss of enzymatic function, 32% did notdemonstrate a significant difference in activity relative to KumaWT, and55% resulted in an increase in observed activity against this substrate.Twenty-eight of the most promising enzyme variants that exhibited asignificant increase in activity in cell lysates were then purified inorder to obtain an accurate comparison of enzymatic activity tothat ofKumaWT. After purification and correction for protein concentration, theactivities of these enzymes ranged from 2-fold to 120-fold more activethan KumaWT (Table 1). The most active variant, which was namesKumaMax™, was selected for further characterization.

TABLE 1 Fold change in hydrolytic activity on PQ motif of all purifiedand sequenced mutants, relative to wild type Kumamolysin-As. These arethe fold-change results (calculated as described in SupplementaryTable 1) for all mutants that were purified, sequenced, and testedagainst wild-type Kumamolysin in the pure protein assay. The assay tookplace at pH 4, with enzyme final concentration of 0.0125 mg/mL andsubstrate concentration of 5 μM. Fold Change in Activity of PQHydrolysis Relative to Mutations to Wild Type Kumamolysin-As Wild Type(Preprocessed) Kumamolysin-As Wild Type (WT) 1.0 T357A 2.0 G319S, D368S2.0 D358G 3.0 D293A 3.0 D358N 4.0 G319S, S354N, D358G, D368H 5.0 D358G,D368H 6.0 G319S, D358G, D368H 7.0 N291D, Q361D 7.5 S354N, D358G, D368H9.0 N291D 10.0 N291D, D293A, Q361D, D358N 14.8 N291D, D293A 15.0 N291D,D293A, D358G, Q361D 15.0 N291D, D358N 18.9 N291D, Q361D, D358G 20.0N291D, G319S, D358G, Q361D, D368H 23.1 N291D, D293A, D358N 24.0 S262G,T292S, N291D, G319S, D358G, D368H 29.0 N291D, D293A, G319S, D358G,Q361D, D368H 40.9 T292S, N291D, G319S, D358G, D368H 49.0 N291D, G319S,S354N, D358G, Q361D, D368H 50.0 N291D, G319S, S354N, D358G, D368H 54.6N291D, D293A, G319S, S354N, D358G, Q361D, 58.0 D368H D293T, N291D,G319S, D358G, D368H 58.0 S262K, D293N, N291D, G319S, D358G, D368H 62.0N291D, G319S, D358G, D368H 93.0 V119D, S262K, D293T, N291D, G319S,D358G, 120.0 D368H

KumaMax™ contains seven mutations from the wild-type amino acidsequence: V119D, S262K, N291D, D293T, G319S, D358G, D368H (FIG. 2B). Ofthese, the mutations G319S, D358G, and D368H appear to synergisticallyintroduce a new hydrogen bond with the desired glutamine residue atposition P1. As modeled, the G319S mutation appears to introduce ahydroxyl group that is located 2.5Å from the carbonyl oxygen of theglutamine amide, potentially contributing a new hydrogen bond thatinteracts with glutamine in the P1 pocket. The D368H mutation ispredicted to stabilize the serine hydroxyl, and its position in theactive site is in turn sterically allowed by the D358G mutation. Inaddition to providing a novel desired interaction with glutamine asmodeled, these three mutations also remove the two acidic residuespredicted to stabilize the positively charged arginine residue in thenative KumaWT substrate (FIG. 2). V119D, which was unexpectedlyincorporated during site directed mutagenesis, is located in thepropeptide domain and therefore does not affect catalytic activity ofthe mature enzyme. The other three mutations do not make direct contactswith residues in the P2 . . . P2′ pockets, and therefore likelyintroduce interactions with other components of the hexapeptide, thefluorophore, or the quencher. It is clear that these mutations areimportant for the overall catalytic enhancement observed, as theG319S/D358G/D368H triple mutant alone demonstrated only a 7-foldincrease in catalytic activity over KumaWT: roughly 17-fold lower thanthat determined for KumaMax™.

Kinetic Characterization and Substrate Specificity

The catalytic efficiencies for KumaMax™ and KumaWT against the FQimmunogenic gluten substrate analogue, as calculated by fitting velocityversus substrate curve over 6-100 μM substrate, were found to be 568M⁻¹s⁻¹ and 4.9 M⁻¹s⁻¹, respectively (Table 2). These values areconsistent with the observation that KumaMax™ demonstrated a 120-foldincrease in enzymatic activity towards the FQ substrate in the initialactivity screen mentioned above. Unfortunately, no significantsaturation of velocity at these substrate concentration was observed,and therefore the individual kinetic constants k_(est) and K_(M) couldnot be determined. This is not surprising since previous analyses of thekinetic constants of KumaWT report a Km of 40 μM. Therefore, nosignificant saturation would be expected at substrate oncentrations lessthan 100 μM.

TABLE 2 Kinetic Constants of peptide substrates for KumaMax ™ andKumaWT. The catalytic efficiency (k_(cat)/K_(M) M⁻¹s⁻¹) for both KumaWTand KumaMa ™ for the fluorescently (Fl) quenched (Qu) PQPQLP (SEQ ID NO:34) peptide was fit to a linear curve as no saturation was observed upto 100 μM substrate. The fluorescence signal was quantified as describedin Materials and Methods with a standard curve that accounted forsubstrate quenching of product fluorescence. The catalytic efficiencyfor the pNA-linked peptides was determined in a similar manner, and isdescribed in the Materials and Methods. All fits had at least sixindependently measured rates with an R² greater than 0.9. CatalyticEfficiency M⁻¹s⁻¹ Qu-PQPQLP-Fl Suc-APQ-pNA Suc-APR-pNA Suc-APE-pNASuc-AQP-pNA KumaWT  4.9 ± 0.2 n.d. 131.8 ± 3.8 4.0 ± 0.1 n.d. KumaMax ™568.5 ± 14.6 6.7 ± 0.4 n.d. 1.4 ± 0.2 n.d. n.d. not detected.

While the increased activity of KumaMax™ compared to KumaWT against thefluorescently quenched PQPQLP (SEQ ID NO:34) hexapeptide substratesuggests that KumaMax has increased preference for a PQ dipeptide motif,it does not report directly on substrate specificity. To confirm thatthe specificity of KumaMax™ had indeed been altered to prefer the PQdipeptide over the native PR dipeptide of KumaWT, four peptides in theform of Succinyl-Alanine-P2-P1-P1′ were provided as substrates to bothenzymes in order to assess P2 and P1 specificity. These peptidescontained the reporter p-nitroaniline (pNA) at the P1′ position, whichallows for a spectrometric readout of peptide cleavage. The fourpeptides harbored the following amino acids at the P2 and P1 positions,respectively: proline-glutamine (PQ), proline-arginine (PR),glutamine-proline (QP), and proline-glutamate (PE). Catalyticefficiencies were calculated for each substrate and are reported inTable 2. As in the determination of catalytic activities against the FQsubstrate, no saturation of activity on these peptides by KumaWT orKumaMax™ was observed. This suggests that the pNA group may partiallydisrupt binding in the P1′ pocket, since substrate concentrations up to1 mM were tested, well beyond saturation levels previously reported foralternative KumaWT substrates.

In this specificity assay, KumaMax™ demonstrated its highest level ofactivity on the PQ substrate, the dipeptide that it had been designed toprefer. While KumaMax™ was not explicitly designed to demonstrate adecrease in specificity for the PR motif or for other motifs, itsincreased specificity for PQ could decrease its activity fornon-targeted motifs. Indeed, KumaMax™ exhibited no significant catalyticactivity against the QP or PR substrates in this assay (Table 2).Consistent with previous reports¹⁴, KumaWT exhibited its highest levelof activity on the PR motif. KumaWT demonstrated significantly lowerlevels of activity on the three other peptide substrates. Whilecatalytic activity of KumaWT on the PQ dipeptide motif has previouslybeen reported¹⁴, no significant activity on the PQ dipeptide substratewas observed in this assay, which may be due to disruptive effects ofpNA on the binding of this peptide to the enzyme active site. Bothenzymes demonstrated activity towards the isosteric substrate PE, whichispredicted tohave neutral charge at pH 4; however, KumaMax™demonstrated a roughly 5-fold decrease in activity on the PE peptidesubstrate compared to its activity on the PQ substrate, whichillustrates its exquisite selectivity for the PQ dipeptide motif.

As discussed previously, there are several enzymes currently beingexplored as OET for Celiac disease. Two of these enzymes are engineeredforms of the prolyl endopeptidase SC PEP and the glutamine-specificendoprotease EP-B2¹⁵. To compare the catalytic efficiencies of theseproteases to that of KumaMax™, the native SC PEP and EP-B2 enzymes wereexpressed in E. coli BL21(DE3) cells, purified, and their catalyticactivities assessed. SC-PEP demonstrated a catalytic efficiency of 1.6M⁻¹s⁻¹ on the FQ gluten substrate analogue at pH 4, which represents aroughly 350-fold lower level of activity on this substrate thanKumaMax™. At pH 4, SC PEP did not exhibit any significant activity onany of the four pNA linked peptide substrates, including QP. Althoughprevious studies using similar pNA-linked peptides have demonstratedactivity of SC PEP on these substrates, those assays were performed at apH of 4.5 or higher¹². Like other groups, we found that SC PEPdemonstrated significant levels of activity on the QP substrate at pH 7,with a catalytic efficiency of 2390 M⁻¹s⁻¹, thereby confirming that thisrecombinant SC PEP was fully functional (data not shown). This isconsistent with previous literature reporting that SC PEP has low tonegligible levels of catalytic activity in the pH range of the stomach,and is thus only expected to be effective once α-gliadin peptides havereached the small intestine^(12,16).

For EP-B2, only very low levels of activity were detected on the FQsubstrate at pH 4, and no activity on any of the four pNA peptidesubstrates were observed (data not shown). This is inconsistent withprevious reports of EP-B2 activity using comparable substrates¹³, EP-B2is a difficult enzyme to purify, as it forms inclusion bodies in E. coliand requires refolding to obtain active enzyme. We were unable to obtainsoluble protein using previously reported methods for the refolding ofEP-B2^(13,12,35), so we used an on-column refolding process whichresulted in soluble protein produced. Although this soluble EP-B2demonstrated the expected self-processing activity at pH 4¹¹ (data notshown), the lack of activity of this enzyme suggests that it may nothave refolded properly using our methods. This could be due toalternative N and C-terminal tags arising from the use of differentprotein expression vectors and warrants for further investigation.

Protease Stability

In addition to demonstrating catalytic activity at low pH, any proteintherapeutic intended for use in the human digestive tract must exhibitresistance to degradation by digestive proteases. Two of the mostabundant proteases in the stomach and small intestine are pepsin andtrypsin, respectively. Pepsin demonstrates optimal proteolytic activityat the low pH ranges of the stomach, while trypsin is primarily activeat the more neutral pH of the small intestine. To assess the resistanceof KumaMax™ to degradation by these proteases, 0.01 or 0.1 mg/mL ofKumaMax™ were incubated with each protease, in their respective optimalpH ranges, at 0.1 mg/mL, which is a physiologically relevantconcentration of both pepsin and trypsin. SC PEP and EO-B2 were includedas controls, as EP-B2 has been established to be resistant to pepsin butsusceptible to trypsin, and SC PEP demonstrates susceptibility to bothproteases^(11,15). Each protein was incubated in the presence or absenceof the respective protease for 30 minutes, after which the proteins wereheat inactivated and the remaining non-proteolyzed fraction determinedusing an SDS-PAGE gel (FIG. 3).

In this assay, KumaMax™ demonstrated high stability against both pepsinand trysin, with roughly 90% intact protein remaining after the halfhour incubation with either protease (FIG. 3). Consistent with previousreports, SC PEP exhibited susceptibility to both pepsin and trypsin,with less than 20% of the enzyme remaining after incubation with theseproteases. As expected, trypsin efficiently proteolyzed EP-B2 with lessthan 10% remaining after incubation, but no significant degradation ofEP-B2 was observed in the presence of pepsin. To confirm that observedprotein degradation was due to protease activity and not to enzymaticself-processing, each enzyme was incubated at either pH 4 or 7 andapparent proteolysis was analyzed in the absence of other proteases overthe course of an hour (data not shown). KumaMax™ and EP-2, but not SCPEP, demonstrated self-processing from the pro-peptide to the activeenzyme form in fewer than 10 minuses at pH 4. All three proteinsremained >90% stable over the course of the hour. None of these proteinsshowed significant levels of self-processing or proteolysis duringincubation for one hour at pH 7.

Degradation of an Immunogenic α9-Gliadin Peptide

The significant level of catalytic activity exhibited by KumaMax™ onimmunogenic peptide analogies (Table 2) demonstrates promise for the useof KumaMax™ as a therapeutic in OET for gluten intolerance. However,these assays do not directly assess the ability of this enzyme todegrade relevant immunogenic peptides derived from gluten. Therefore, weexamined the direct proteolytic activity of KumaMax™ towards animmunodominant peptide present in α9-gliadin, QLQPFPQPQLPY (SEQ IDNO:70).

KumaMax was incubated with 500 μM of the α9-gliadin peptide at 37° C. inpH 4 at roughly a 1:100 enzyme to peptide molar ratio, which representsa physiologically relevant concentration of this peptide in the humanstomach. SC PEP was included in this experiment for the sake ofcomparison, since this enzyme demonstrates significantly less activityagainst the FQ substrate than KumaMax™. Samples from the incubation werequenched every 10 minutes in 80% acetonitrile to halt the proteolysisreaction. The remaining fraction of intact immunogenic peptide wasdetermined using high-performance liquid-chromatography massspectroscopy, in which the M+H parent ion of the α9-gliadin peptide wasmonitored. KumaMax™ demonstrated a high level of activity against theimmunogenic peptide in this assay, as over 95% of the immunogenicpeptide had been proteolyzed alter a 50 minute incubation with KumaMax™,while no significant degradation of the peptide was observed in thepresence of SC PEP or in the absence of protease (FIG. 4A). Thehalf-life of the peptide in the presence of KumaMax™ was determined byplotting the fraction of peptide remaining against the incubation time,and was calculated to be 8.5±0.7 minutes (FIG. 4B).

Discussion

Enzyme therapy is an attractive method for the treatment of Celiacdisease since this form of treatment would not require intravenousinjection. However, it is a challenge to identify an appropriateprotease for use in OET that demonstrates all the properties necessaryto be an effective therapeutic for Celiac disease. Specifically, anideal protease for use in OET would maintain activity in a pH range from2-4 at 37° C. and would resist degradation by common digestiveproteases. In addition, the protein therapeutic would ideallydemonstrate stringent specificity for a common motif found inimmunogenic gluten-derived peptides. Finally, the protein should beeasily produced using recombinant methods. While it is unlikely that asingle natural enzyme will encompass all of these properties, wedemonstrate that a protein containing several of these importantcharacteristics can be engineered to demonstrate the lacking qualitiesthrough computational analysis, mutagenesis, and screening.

The engineered protease, KumaMax™, demonstrated a high level of activityon, and specificity towards, the desired PQ dipeptide motif (Table 2).The specificity for the PQ motif, as opposed to the native PR motif,potentially derives from the addition of new hydrogen bonds in the S1pocket of KumaMax that, as modeled, make direct contacts with theglutamine in this dipeptide motif (FIG. 2B). This specificity switch notonly directs activity against a motif found commonly throughout gluten,but it also greatly decreases activity against non-targeted substrates(Table 2). The inability for an oral protease to recognize non-targetedsubstrates is an important characteristic as it reduces competitiveinhibition by the large number of other peptides produced in the stomachduring digestion of a meal. KumaMax™ or KumaWT can potentially act asplatforms for engineering greater specificity, as KumaWT hasdemonstrated some level of selectivity beyond the P2 and P1 sites¹³.Using this method, a panel of customized proteases specific for uniqueimmunogenic epitopes could be generated.

Methods Protein Expression and Purification

The genes encoding each protein of interest, harbored in the pET29bplasmid, were transformed into Escherichia coli BL21 (DE3) cells.Individual colonies were picked, inoculated into Terrific Broth™ with 50μg/μL Kanamycin (TB+Kan), and incubated overnight at 37° C. 500 uL ofthe overnight culture was added to 500 mL autoinduction media (5 gtryptone, 2.5 g yeast extract, 465 mL ddH₂O), and shaken at 37° C. forroughly 4 hours, then the autoinduction components were added (500 uLMgSO₄, 500 uL 1000x trace metals. 25 mL 20x NFS, 10 mL 20x 5052, 500 uL50 mg/mL Kan). The culture were then shaken at 18° C. for 30 hoursbefore being spun down. Pellets were resuspended in 10 mL 1x PBS, thenlysed via sonication with 5 mL lysis buffer (50 mM HEPES, 500 mM NaCl, 1mM bMF, 2 mg/mL lysozyme, 0.2 mg/mL DNase, ddB₂O) and spun down. Theproteins were then purified over 1 mL TALON cobalt affinity columns.KumaMax. KumaWT, and SC Pep were washed three times with 20 mL washbuffer (10 mM imidazole, 50 mM HEPES, 500 mM NaCl, 1 mM bME, ddH₂O), andthen eluted in 15 mL of elution buffer (200 mM imidazole, 50 mM HEPES,500 mM NaCl, 1 mM bME). EP-B2 had to be refolded on the column, so afterlysis the pellets were resuspended in 10 mL of EP-B2 buffer, whichdiffers from the wash buffer only in that it is diluted in guanidinehydrochloride instead of ddH₂O to allow for denaturation of the EP-B2inclusion bodies. This resuspension was pelleted, and the supernatant(containing denatured PP-B2) was filtered with a 0.8 μm filter onto thecolumn. EP-B2 was washed once with 20 mL of the BP-B2 buffer, beforebeing washed twice with 20 mL of the wash buffer to refold the proteinon the column. Protein was eluted with 15 ml of the elution buffer. Allproteins were concentrated from 15 mL down to ˜500 uL, then dialyzedonce in 1 L dialysis buffer (20% glycerol, 50 mM HEPES, 500 mM NaCl, 1mM bME). Protein concentration was calculated spectrophotometricallywith extinction coefficients of 53,985 M⁻¹cm⁻¹ for KumaWT and all KumaWTvariants, 152,290 M⁻¹cm⁻¹ for SC Pep, and 58,245 M⁻¹cm⁻¹ for EP-B2.

Screening Method

Kunkel mutagenesis was used to generate mutations to KumaWT. Individualcolonies picked from plates were grown up in 90-deep well plates. Afterlysing the cells with Triton buffer (1% 100X Triton, 1 mg/mL lysozyme,0.5 mg/mL DNase, 1x PBS), the supernatant was adjusted to pH 4 with a100 mM sodium acetate buffer. To crudely screen for activity against theFQ substrate, 10 μL of supernatant was added to 90 uL of 5 μM substratesin a 96-well black plate, and the fluorescence was measured at 30-secondintervals for 1 hour.

Purified Enzyme Assay

The variants of Kumamolisin-As that displayed the most activity on theFQ substrate in the activity screen, were sequenced, then purified insmall scale. 500 uL of TB+Kan overnight cultures were added to 50 mLTB+Kan and grown at 37° C. until reaching an optical density of 0.5-0.8.OPTO was added to 0.5 mM, and the cultures were expressed at 22° C. for16-24 hours. The cells were spun down, resuspended in 500 uL of washbuffer (1s PBS, 5 nM imidazole, ddH₂O), transferred to a 2 mL Eppendorftube, and lysed in 1 mL lysis buffer (1x PBS, 5 mM imidazole, 2x BugBuster™, 2 mg/mL lysozyme, 0.2 mg/mL. DNase, ddH₂O). Aftercentrifugation, the supernatant was decanted into a fresh tube. Columnswith 200 uL of TALON cobalt resin were placed in Eppendorf tubes, andthe supernatant was poured over the columns and rocked for 20 minutesbefore spinning down and discarding the flow-through. The proteins werewashed three times with 500 uL wash buffer, discarding the flow-throughbetween washes. Enzymes were eluted in 200 uL elution buffer (1x PBS,200 mM imidazole, ddH₂O), and concentrations were calculatedspectrophotometrically using an extinction coefficient of 53,985M⁻⁴cm⁻³.

For the assay, the Kumamolisin-As mutants were incubated for 15 minutesin pH 4 100 mM sodium acetate buffer. Enzyme was added to 5 μM substrateso that the final protein concentration was 0.0125 mg/mL. Thefluorescence was measured at 30-second intervals for 1 hour.

Kinetic Characterization

Enzyme variant proclivity for gluten degradation was measured byhydrolysis of the fluorescently quenched α-gliadin hexapeptide anaogueQXL520-PQPQLP-K(5-FAM)-NH2 (FQ) (SEQ ID NO:69) as a substrate. Eachenzyme was incubated at room temperature for 15 minutes in 100 mM pH 4sodium acetate buffer. After 15 minutes, 50 uL of fluorescent substratewas added ranging in final concentration between 100, 50, 25, 12.5,6.25, and 0 μM peptide, and maintaining concentrations of 0.05 μMKumaMax™, 0.5 μM KumaWT 0.5 μM SC Pep, and 0.5 μM EP-B2 across allvariations in substrate concentration. The plate was read immediately onthe spectrophotometer for an hour, using 455 nm wavelength forexcitation and reading 485 nm wavelength for emission.

The enzymes were also tested for specificity to different dipeptidemotifs using a variety of chromogenic substrates that releasep-nitroaniline (pNA) upon hydrolysis: [Suc-APQ-pNA], [Suc-AQP-pNA],[Suc-APE-pNA], and [Suc-APR-pNA]. Again, each enzyme was incubated atroom temperature for 15 minutes in 100 mM pH 4 sodium acetate buffer.After 15 minutes, 20 uL of substrate was added to the enzyme incubationso that the final concentrations of substrate ranged between 1000, 500,250, 125, 62.5, 31.25, 15.625, and 0 μM, and all enzymes being testedended in a concentration of 0.5 μM. The plate was read immediately onthe spectrophotometer for an hour, monitoring absorption by thereactions at 385 nm.

The standard curve for the fluorescent peptide involved mixing substrateand product together at varying concentrations in pH 4 buffer. Substrateconcentrations were 100, 50, 25, 12.5, 6.25, and 0 μM, and productconcentrations were 20, 1.25, 0.3125, 0.078125, 0 μM.

The standard curve for the absorbent peptide involved productconcentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125,0.390625, 0.1953125, 0.09765625, and 0 μM dilated in pH 4 buffer.

Protease Stability

Enzymestability was determined in the presence the digestive proteases,pepsin and trypsin KumaWT, KumaMax™, SC Pep, and EP-B2 were incubated inbuffer matching the native pH environment of each digestive protease, Ph3.5 100 mM sodium acetate was used to pre-incubate the enzymes forpepsin digestion assays, and pH 7.5 dialysis buffer (see “ProteinExpressiona and Purification”) for trypsin digestion assays. Eachexperimental enzyme was incubate at 37° C. for 15 minutes in eachbuffer, at a concentration of 0.2 mg/mL.

After pre-incubation in the appropriate buffer, 0.1 mg/mL digestiveprotease was added. The reactions were done in triplicate, and wereincubated at 37° C. for 30 minutes. Adding SDS and boiling for 5 minutesensured digestive protease inactivation. An SDS-PAGE gel allowedquantification of enzyme degradation, using ImageJ.

The rate of protein self-proteolysis was determined at pH 4 ad 7.5 inthe absence of pepsin or trypsin. Each enzyme, at a concentration of 0.2mg/mL, was incubated in pH 4 100 mM sodium acetate and pH 7.5 dialysisbuffer. At 20, 40, and 60 minutes, timepoints were taken. SDS wasadded,a nd the aliquots were boiled for 5 minutes to ensure denaturationo the enzymes and inhibition of further self-proteolysis. Again, anSDS-PAGE gek in conjunction with ImageJ allowed quantification of enzymeself-proteolysis.

LCMS Gliadin Degradation Assay

Enzyme activity on full-length α0gliadin was measured usinghigh-performance liquid-chromatography mass spectrometry. For eachenzyme, 7 μL of pH 4 1M sodium acetate buffer was added to 28 μL of 5 μMenzyme, and incubated alongside separate tubes of 3 μL gliadin at 37° C.for 15 Minutes Next 27 μL of each enzyme mixture, and 27 μL of dialysisbuffer as a control, were added to each tube of gliadin. These wereincubated once

more at 37″ C, and 5 μL samples were taken at 10, 20, 30, 40, and 50minutes. Each timepoint sample was quenched in 95 μL of 80% acetonitrilewith 1% formic acid and approximately 33 μM leupeptin. The samples wereanalyzed on the HPLC to compare gliadin degradation by the differentproteases over time.

REFERENCES

1. Armstrong, M. J., Hegade, V. S. Robins, G. Advances in coeliacdisease. Curr Opin Gastroenterol 28, 104-12 (2012).

2. Sollid, L. M. Coeliac disease; dissecting a complex inflammatorydisorder. Nat Rev Immunol 2, 647-55 (2002).

3. Wieser, H. Chemistry of gluten proteins. Food Microbiol 24, 115-9(2007).

4. Shan, L, et al. Structural basis for gluten intolerance in celiacsprue. Science 297, 2275-9 (2002).

5. Shan, L, Identification and analysis of multivalent proteolyticallyresistant peptides fro gluten: implications for celiac sprue. Journal ofProteome Research (2005).

6. Chand, N. & Mihas, A. A. Celiac disease: current concepts indiagnosis and treatment J Clin Gastroenterol 40, 3-14 (2006).

7. Shan, L., Marti, T., Sollid, L. M., Gray, G. M. & Khosla, C.Comparative biochemical analysis of three bacterial prolylendopeptidases: implications for coeliac sprue, Biochem J 383, 311-8(2004).

8. Siegel, M. et al. Rational design of combination enzyme therapy forceliac sprue. Chem Biol 13, 649-58 (2006).

9. Stepniak, D. et al. Highly efficient gluten degradation with a newlyidentified prolyl endoprotease: implications for celiac disease, Am JPhysiol Gastrointest Liver Physiol 291, G621-9 (2006.)

10. Ehren, J. et al. A food-grade enzyme preparation with modest glutendetoxification properties, PloS One 4, e6313 (2009).

11. Bethune, M. T., Strop, P., Tang, Y., Sollid, L. M. & Khosla, C.Heterologous expression, purification, refolding, andstructural-functional characterization of EP-B2, a self-activatingbarley cysteine endoprotease. Chem Biol 13, 637-47 (2006).

12. Okubo, A. et al. Processing, catalytic activity and crystalstructures of kumamolisin-As with an engineered active site. FEBS J 273,2563-76 (2006).

13. Gardner, J. D., Ciociola, A. A. & Robinson, M. Measurement ofmeal-stimulated gastric acid secretion by in vivo gastric autotitration,J Appl Physiol 92, 427-34 (2002).

14. Wlodawer, A. et al. Crystallographic and biochemical investigationsof kumamolisin-As, a serine-carboxyl peptidase with collagenaseactivity, J Biol Chem 279, 21500-10 (2004).

15. Ehron, J., Govindarajan, S., Moron, B. Minshull, J. & Khosla, C.Protein engineering of improved prolyl endoeptidases for celiac spruetherapy, Protein Eng Des Sel 21, 699-707 (2008).

16. Bethune, M. T. & Khosla, C. Oral enzyme therapy for celiac sprue.Methods Enzymol 502, 241-71 (2012).

17. Gass, J., Vera, H., Bethune, M. T., Gray, G. M. & Khosla, C. Effectof barley endoprotease EP-B2 on gluten digestion in the intact rat. JPharmacol Exp Ther 318, 1178-86 (2006).

18. Vera, H., McIntire, J., Kumar, P., Deshpande, M. & Khosla, C. Ascaleable manufacturing process for pro-EP-B2, a cysteine protease frombarley indicated for celiac sprue Biotechnol Bioeng 98, 177-85 (2007).

1.-20. (canceled)
 21. A polypeptide comprising an amino acid sequence atleast 75% identical to the amino acid sequence of SEQ ID NO:1 residues1-567, wherein (a) the polypeptide degrades a PQPQLP (SEQ ID NO:34)peptide at pH 4; (b) residue 467 is Ser, residue 267 is Glu, and residue271 is Asp; and (c) the polypeptide comprises one or more amino acidchanges from SEQ ID NO: 33 at one or more residues selected from thegroup consisting of amino acid residues 119, 262, 291, 292, 293, 319,354, 357, 358, 361, and 368, wherein if the amino acid change is atresidue 262, the residue 262 amino acid is changed to either K or G. 22.The polypeptide of claim 21, comprising an amino acid sequence at least85% identical to the amino acid sequence of SEQ ID NO:1 residues 1-567.23. The polypeptide of claim 21, comprising an amino acid sequence atleast 95% identical to the amino acid sequence of SEQ ID NO:1 residues1-567.
 24. The polypeptide of claim 21, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:1 residues 1-567.
 25. Thepolypeptide of claim 21, wherein the polypeptide comprises the aminoacid sequence of any one of SEQ ID NO:2-32 residues 1-567.
 26. Thepolypeptide of claim 21, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:32 residues 1-567.
 27. The polypeptide ofclaim 21, wherein the one or more amino acid changes are selected fromthe group consisting of: Residue 119: D; Residue 262: K and G; Residue291: D; Residue 292: S; Residue 293: A, T, and N; Residue 319: S;Residue 354: N; Residue 357: T; Residue 358: N and G; Residue 361: Q;and Residue 368: S and H.
 28. A nucleic acid encoding the polypeptide ofclaim
 21. 29. A nucleic acid expression vector comprising the isolatednucleic acid of claim
 28. 30. A recombinant host cell comprising thenucleic acid expression vector of claim
 29. 31. A pharmaceuticalcomposition, comprising the polypeptide of claim 21 and apharmaceutically acceptable carrier.
 32. A method for treating celiacsprue, comprising administering to an individual with celiac sprue anamount effective to treat the celiac sprue of the polypeptide of claim21.
 33. The method of claim 32, wherein the polypeptide comprises anamino acid sequence at least 85% identical to the amino acid sequence ofSEQ ID NO: 1 residues 1-567.
 32. The method of claim 32, wherein thepolypeptide comprises an amino acid sequence at least 95% identical tothe amino acid sequence of SEQ ID NO: 1 residues 1-567.
 35. The methodof claim 32, wherein the polypeptide comprises the amino acid sequenceof SEQ ID NO:1 residues 1-567.
 36. The method of claim 32, wherein thepolypeptide comprises the amino acid sequence of any one of SEQ IDNO:2-32 residues 1-567.
 37. The method of claim 32, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:32 residues1-567.
 38. The method of claim 32, wherein the one or more amino acidchanges are selected from the group consisting of: Residue 119: D;Residue 262: K and G; Residue 291: D; Residue 292: S; Residue 293: A, T,and N; Residue 319: S; Residue 354: N; Residue 357: T; Residue 358: Nand G; Residue 361: Q; and Residue 368: S and H
 39. A method fortreating celiac sprue, comprising administering to an individual withceliac sprue an amount effective of the pharmaceutical composition ofclaim 31 to treat the celiac sprue.
 40. The method of claim 32, whereinthe polypeptide is administered orally.